GM130对胃癌细胞O-糖基化及细胞粘附能力的影响

Effect of GM130 on O-linked glycosylation and adhesion of human gastric cancer cell line MKN-45

目的:初步探讨沉默高尔基体基质蛋白130(golgi matrix protein 130,GM130)基因对胃癌细胞MKN-45 O-糖基化及细胞粘附能力的影响。方法:通过si RNA干扰胃癌细胞中GM130的表达水平,用Lipofectamine 2000将GM130-si RNA转染入MKN-45细胞。实验分为GM130-si RNA组、阴性对照组和空白对照组。分别用q RT-PCR和Western blot检测转染后各组中GM130、N-乙酰氨基半乳糖转移酶2(polypeptide N-acetylgalactosaminyl transferase 2,pp Gal NAc-T2)、核心1β3半乳糖基转移酶(core 1 synthase,glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase1,C1Gal-T1)、β-半乳糖α-2,3-唾液酸转移酶1(beta-galactoside alpha-2,3-sialyltransferase,ST3Gal-1)m RNA和蛋白表达的变化。应用凝集素流式细胞术检测3组细胞中α2,3唾液酸残基糖链结构的表达水平。肿瘤细胞与内皮细胞粘附实验及基质胶粘附实验检测细胞粘附能力。结果:q RT-PCR和Western blot结果显示GM130基因与蛋白水平明显受到抑制,表明转染成功;GM130-Si RNA组C1Gal-T1、ST3Gal-1的m RNA和蛋白表达水平均明显降低(P均<0.05),pp Gal NAc-T2在3组中无明显变化(P>0.05),α2,3唾液酸糖链结构的表达量降低(P<0.05),细胞粘附能力下降(P<0.05)。结论:在MKN-45细胞中,下调GM130后,可能通过下调C1Gal-T1、ST3Gal-1的表达影响胃癌细胞O-糖基化,降低肿瘤细胞粘附能力。

Objective：To preliminarily investigate the effect of RNA interference targeting GM130 on O-linked glycosylation and adhesion of human gastric cancer cell line MKN-45. Methods：GM130-si RNA was transfected into human gastric cancer cell line MKN-45 with Lipofectamine 2000. The experiment had three groups ：GM130-si RNA group,negative control group and blanck control group. The expressions of GM130,pp Gal NAc-T2,C1Gal-T1,ST3Gal-1 at m RNA and protein levels in the MKN-45 cells were analyzed by real-time PCR and Western blot. The expressions of α2,3-linked sialic acid residues in MKN-45 cell surface were detected by flow cytometry. The changes in the adhesion of the transfected cells were tested with matrigel adhesion and tumor cell adhere to the vascular endothelial cell assay. Results：The real-time PCR and Western blot results showed that the m RNA and protein expression levels of GM130 decreased significantly in gastric cancer cell line MKN-45 transfected with GM130-si RNA,indicating a successful transfection. Compared with those in blank and negative control groups,both the m RNA transcription and protein expression levels of C1Gal-T1,ST3Gal-1 in MKN-45 cells transfected with GM130-si RNA decreased significantly（P〈0.05）,but those of pp Gal NAcT2 were not significantly affected（P〈0.05）. The expression levels of α2,3-linked sialic acid residues were decreased（P〈0.05）,and the cell adhesion was apparently reduced（P〈0.05）. Conclusion：The GM130 may play an important role in O-linked glycosylation of MKN-45;the molecular mechanism might be associated with down-regulating C1Gal-T1,ST3Gal-1 expression and downregulation of GM130 decreasd gastric cancer cell adhesion.